Effects of long-term lamivudine therapy in renal-transplant patients

Journal of Clinical Virology 31 (2004) 298–303

Effects of long-term lamivudine therapy in renal-transplant patients Nassim Kamara , Karine Sandres-Sauneb , David Ribesa , Michel Duffautc , Jannick Selvesd , Dominique Duranda , Jacques Izopetb , Lionel Rostainga,∗ a c

Multiorgan Transplant Unit, CHU Rangueil, 1 Avenue J. Poulh`es, TSA 50032, 31059 Toulouse C´edex 9, France b Laboratory of Virology, CHU Purpan, Place du Dr. Baylac, TSA 40031, 31059 Toulouse C´ edex 9, France Department of Internal Medicine, CHU Purpan, Place du Dr. Baylac, TSA 40031, 31059 Toulouse C´edex, France d Department of Pathology, CHU Purpan, Place du Dr. Baylac, TSA 40031, 31059 Toulouse C´ edex, France Received in revised form 1 April 2004; accepted 1 July 2004

Abstract Background : Following renal transplantation (RT), chronic immunosuppression is associated in hepatitis B virus (HBV) (+) patients with a flare-up of the disease, which might be harmful in the long term. Objectives : We report on the effect of long-term lamivudine therapy given at an initial daily dose of 100 mg in 18 HBV (+) RT patients. Results : When lamivudine therapy was commenced, 14 patients (77%) had an increase in their aspartate (AST) and alanine (ALT) aminotransferase levels. During a mean follow-up, under treatment, of 36.5 ± 3.5 months (up to 66 months), 10 patients (55%) had a sustained partial (HBV DNA < 4 × 105 copies/ml) (n = 4) or complete (HBV DNA < 400 copies/ml) (n = 6) virological response. Overall, 12 virological breakthroughs were observed. Of those who were HBe Ag(+) prior to lamivudine therapy (n = 4), one seroconverted to HBe Ab during therapy. At the last follow-up, AST and ALT levels were normal in 13 patients. When liver biopsy was repeated during treatment (n = 15), the virological responders showed a significant decrease in total Knodell score from 10 ± 0.6 to 7 ± 1 (P = 0.04), but no significant change in the stage of fibrosis. Conversely, in those patients with high HBV DNA titers, there were no significant changes in the total Knodell score or in the grade of fibrosis. Conclusion : In conclusion, lamivudine therapy is safe in HBV(+)ve renal-transplant patients. However, even if the full and partial virological response rates are still high (55%) in the long term, relapse or primary non-responses occur. The implementation of alternative efficient strategies is warranted. © 2004 Elsevier B.V. All rights reserved. Keywords: Renal transplantation; Hepatitis B virus; Lamivudine; Liver histology

1. Introduction Hepatitis B virus (HBV) infection is very prevalent worldwide, causing acute and chronic liver disease, with the number of chronically infected individuals estimated to exceed 300 million (Nassal and Schaller, 1996). In the renal population, i.e., hemodialysis and kidney-transplant patients, the prevalence of HBV infection is, at least in western Europe Abbreviations: HBV, hepatitis B virus; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ␥GT, gamma glutamyl transpeptidase; HBe Ag, HBe antigen; HBe Ab, HBe antibody; CsA, cyclosporine A ∗ Corresponding author. Tel.: +33 561322584; fax: +33 561322864. E-mail address: [email protected] (L. Rostaing). 1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2004.07.001

and North America, low, and ranges from a few percent to less than 10% (Najem et al., 1981). However, following renal transplantation, it has been clearly demonstrated that chronic immunosuppression is associated in HBV(+)ve patients with a significant increase in liver-related complications, including liver cirrhosis and hepatocarcinoma, particularly in those coinfected with hepatitis C virus (Mathurin et al., 1999). This results, at least in the long term, in a high proportion of patients dying from HBV-related liver diseases compared with HBV(−)ve renal-transplant patients. This dramatic outcome in HBV(+)ve patients following transplantation is mostly because, during hemodialysis, HBV infection is quiescent, whereas it flares up with immunosuppression. In the latter setting, alpha interferon, the treatment of choice for chronic

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HBV infection (Wong et al., 1993), is not suitable after renal transplantation because it is associated with acute renal failure as well as with acute rejection (Rostaing et al., 1996). More recently, nucleoside-analogue inhibitors have been developed, e.g., lamivudine (3TC) (Luscombe and Locarnini, 1996). Lamivudine monotherapy has been shown to be efficient in suppressing HBV replication, both in immunocompetent (Lai et al., 1998) as well as in liver- (Grellier et al., 1996) and renal-transplant (Rostaing et al., 1997) patients. However, resistance to this agent has been documented and lamivudine-resistant isolate changes are mainly found in both the B domain and the YMDD motif (the C domain) of DNA polymerase (Bartholomeusz et al., 1998). The development of resistance due to mutations might be of great concern, particularly in a population such as transplant patients. We have already reported on the short-term efficacy of lamivudine therapy in suppressing HBV replication in renal-transplant patients; however, we observed a universal relapse after withdrawal of the drug (Rostaing et al., 1997). Our initial results were confirmed by Jung et al. (1998) in a small sample of patients. In this paper, we report on the long-term results of a larger cohort of HBV(+)ve renal-transplant patients treated with lamivudine with a special emphasis on its impact on liver histology. Overall, lamivudine therapy was safe. In the long term, even if many patients experienced a virological relapse, liver histology was at least stable or even improved in the subset of patients.

2. Patients and methods 2.1. Patients Eighteen recipients of cadaveric transplants (14 men, 4 women) with post-transplant reactivation of HBV were included in this open study. Their mean age was 46.5 ± 2.5 years. At the time of the most recent transplantation (which was their first in nine of the cases, and iterative one, i.e. >1, in 9 cases), all were HBs Ag(+), only two were HBe Ag(+) whereas 16 of 18 were HBe Ab(+). They had been on hemodialysis for 108 ± 20 months. In all cases, HBV infection was acquired while on hemodialysis, i.e., 207 ± 18 months prior to lamivudine therapy. Reactivation of HBV infection after renal transplantation, defined as an increase in ALT/AST and/or gamma glutamyl transpeptidase levels, occurred at 12 ± 3 months post-transplantation. Reactivation was associated with the loss of HBe Ab in three patients and the reappearance of HBe Ag in these three. In those who were previously HBe Ag(+), one spontaneously lost the HBeAg and became HBe Ab(+). Therefore, when lamivudine therapy was implemented, serum HBs Ag and HBV DNA were positive in all patients: 14 patients were HBe Ab(+) and only four patients were HBe Ag(+). As has been carried out at our institution since 1986, immunosuppression was sequential and included antilymphocyte preparations in all patients in addition to pred-

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nisolone. Anti-calcineurin agents, i.e., cyclosporine A (CsA) or tacrolimus were given, respectively, to 14 and 2 patients, and this was begun at around day 8 post-transplantation. Moreover, 13 of the 18 patients received, in addition, azathioprine or mycophenolate mofetil. Four patients presented with an episode of acute rejection, and were treated with methylprednisolone pulses (one case) or OKT3 (5 mg/day for 10 consecutive days) therapy (three cases). Within the 3 months prior to starting lamivudine therapy, all the patients underwent a liver biopsy, which was assessed by the same pathologist (J.S.) using the Knodell scale. The liver biopsy was normal in two cases, whereas it showed chronic active hepatitis in 11 cases and liver cirrhosis in five cases. Inclusion criteria for this study were: (i) being a recipient of a renal graft with post-transplantation reactivation of hepatitis B virus, (ii) having had a pre-study liver biopsy, and (iii) having a transient or a persistent increase in ALT levels. None of the patients were co-infected by hepatitis C or hepatitis D virus. As lamivudine has been available for only a few years, the treatment was only begun at 66 ± 15 months post-transplantation (see Table 1). At that time, the mean serum creatinine was 148 ± 16 ␮mol/l. The first patients commenced in 1996. As recommended for the treatment of hepatitis B infection, lamivudine treatment was started at 100 mg daily, orally, in 16 patients. In the last two patients, due to a serum creatinine level greater than 200 ␮mol/l, we started lamivudine at 50 mg per day. Based on the virological parameters obtained at 3 months after beginning lamivudine, if HBV DNA was still present, the daily dose was doubled because the efficacy of lamivudine upon HBV is dose-dependent (Dienstag et al., 1995). When we started the study, in the absence of guidelines, the treatment was scheduled for 6 consecutive months. Because we observed a universal relapse after the withdrawal of lamivudine therapy in the first four patients (Rostaing et al., 1997), from that time onwards, lamivudine monotherapy was given on a longterm basis, even in the occurrence of biological/virological relapses. When breakthrough occurred, i.e. HBV DNA retesting positive while testing negative previously, we increased lamivudine, sometimes up to 300 mg/day. The primary endpoint of the study was the virological response as defined by the negativation of HBV DNA titers (see below). When no virological response occurred or in a setting of virological relapse, we performed a molecular analysis (see below) in order to assess the de-novo mutations in the C domain (YMDD motif) or B domain of HBV DNA polymerase. 2.2. HBV DNA quantitative analysis HBV DNA was quantitatively analyzed at the beginning of the study and then every 3 months. It was quantitatively analyzed by means of the Amplicor HBV MonitorTM test (Roche Diagnostics, Branchburg, USA). The Amplicor HBV Monitor is an in vitro test that utilizes polymerase chain reaction (PCR) nucleic acid amplification and DNA hybridization for

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Table 1 Demographic and biological data of 18 HBV DNA(+) renal-transplant patients treated with lamivudine Patient

Sex age

Time on dialysis (mo)

TR-3 TC (mo)

Knodell score/fibrosis

AST

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

M.51 M.49 F.27 F.59 M.49 M.31 M.50 M.63 F.62 M.51 M.52 M.39 M.28 M.44 M.45 M.47 M.40 F.52

192 160 28 46 13 34 14 8 187 90 24 176 192 186 64 203 222 195

91 20 13 98 119 96 146 10 60 41 64 63 21 29 263 12 63 34

6/1 11/4 9/4 12/4 9/4 9/1 13/3 2/1 2/0 3/0 3/1 5/4 4/0 8/1 10/3 10/3 5/0 5/0

56 170 27 40 53 216 138 33 63 104 33 37 35 95 93 48 96 256

ALT [Day 0] 82 104 48 45 84 578 178 70 145 183 35 51 84 111 204 81 243 361

AST ALT [Follow-up]

3 TC therapy (mo)

54 33 16 40 51 40 25 50 26 23 25 25 59 44 125 37 90 30

43 30 18 23 19 30 30 22 11 31 22 16 22 29 23 41 26 21

84 38 31 43 55 51 27 90 27 37 31 33 107 48 283 49 195 44

Abbreviations: 3TC: lamivudine: TR-3TC: time from renal transplantation to 3TC therapy (months); AST: aspartate aminotransferase (IU/L); ALT: alanine amino transferase (IU/L); LQ: limit of quantification; HBV DNA: hepatitis B virus DNA (log10 copies/ml).

the quantitative measurement of hepatitis B viral DNA in human serum or plasma. The test quantifies virus titers from 4 × 102 to 4 × 107 viral particles per ml of serum or plasma.

test. A P value of ≤0.05 was considered to be statistically significant.

2.3. Detection of resistant HBV strains

3. Results

HBV DNA was extracted from 200 ␮l of serum using a QIAampTM blood and tissue kit (Qiagen, Courtaboeuf, France). Part of the polymerase gene between nucleotides 466 and 863 was amplified by PCR using the primers 5 -GCCCGTTTGTCCTCTAAT-3 and 5 -TAACCCCATCTTTTTGTTTT–3 (Cane et al., 1999) with standard reaction conditions. All the contaminationprevention measures suggested by Kwok and Higushi were strictly applied (Kwok and Higuchi, 1989). The amplification products were sequenced in the sense and antisense directions by the di-deoxy chain termination method (ABI PRISMTM Ready Reaction AmpliTaq Fs, Dye DeoxyTM Terminators, Applied Biosystems, Paris, France) on an ABI 377 automated DNA sequencer (Applied Biosystems). Multiple alignments were done with CLUSTALW version 1.7 (Thompson et al., 1996). Sequences were compared with each other for their control sequences by phylogenetic analysis and distance mapping (Saitou and Nei, 1987) to exclude any possibility of sample contamination or mix up. HBV DNA phylogenies and bootstrap analyses were performed with reference strains using programs from the PHYLIP phylogeny inference package (version 3.5c, by Joseph Felsenstein) to determine HBV genotype (Norder et al., 1994).

To date, the 18 patients have been treated for 36.5 ± 3.5 months; of these, 14 have been on lamivudine therapy for more than 26 months (i.e., up to 66 months; see Table 1). The last four patients died during lamuvidine therapy (two myocardial infarctions; one mesenteric infarction; one traffic accident). The clinical and biological tolerances were very good.

2.4. Statistical analysis All results are expressed as mean ± SE. Comparison of values obtained before and during lamivudine therapy was performed using a non-parametric test, i.e., the Wilcoxon

3.1. Virological parameters 3.1.1. HBV DNA titers At the last follow-up, HBV DNA quantitative analysis was negative in six patients (complete responders), i.e., <400 copies/ml achieved by daily doses ranging from 100 to 300 mg/day. In four cases, the HBV DNA titer was quite low, i.e., <4 × 105 copies/ml (partial responders), but not negative despite daily doses of lamivudine ranging from 150 to 200 mg. In the last eight patients, no response (n = 1) or relapse after the initial virological response (n = 7) was observed, despite increasing the daily dosage of lamivudine up to 300 mg/day (non-responders). After lamivudine therapy was commenced, HBV DNA titers decreased significantly in both the (complete and partial) responders and the non-responders, i.e., from 8.2 ± 0.4 log copies/ml to less than 2.6 log copies/ml (P = 0.0001) and from 8.3 ± 0.4 log copies/ml to 6.6 ± 0.6 log copies/ml (P = 0.024), respectively. However, HBV DNA titers at the beginning of lamivudine therapy were not statistically different

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between the (complete and partial) responders and the nonresponders. Of the four patients who were HBe Ag(+) at the start of lamivudine therapy, only one became HBe Ab(+) during the treatment. 3.1.2. Virological breakthrough during lamivudine therapy Overall, 7 virological breakthroughs were observed. Initially, in these 7 patients, HBV DNA became negative; however, it returned to pre-treatment baseline values in only five patients, whereas in the last two patients, HBV DNA viremia recurrence remained at low levels (see Table 2). In every patient, breakthroughs were associated with either a transient or a permanent increase in ALT levels. 3.1.3. HBV DNA polymerase mutations All the patients were of genotype D. Amongst the 12 patients testing positive HBV DNA at last follow-up, we were able to assess HBV DNA mutations in the B and C domains in nine patients: no samples were available for the last three patients (see Table 2). Patient 1 who did not respond to lamivudine had no mutation. Amongst the seven patients with virological breakthrough during therapy, results were available for five; of these, three patients showed mutations at both L526 and M550 positions. Among the four patients with a partial virological response, three were assessed for HBV DNA polymerase mutations: two showed mutations.

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normal in all but four patients, i.e., cirrhotic patients. After initiation of the treatment, there was no flare-up of liver enzymes. At 1 month after therapy commenced, AST and ALT were normalized in only four of the 14 patients who had initially increased liver enzymes. At last follow-up, aminotransferase levels were normal in 13 patients, i.e., they were normalized in nine patients and were already normal at the beginning of lamivudine therapy in the other four patients, and still increased in five (see Table 1). 3.3. Follow-up liver biopsies Of the 18 patients followed, 15 had liver biopsies at 18–24 months after the beginning lamivudine therapy. Of these, five (33.3%) were responders, i.e., HBV DNA was negative by a quantitative test at last follow-up. Overall, there was no change in either the stage of fibrosis (1.9 ± 0.4 before and after) or in the total Knodell (7.6 ± 0.9 before and 6.1 ± 0.9 after) and the activity (5.6 ± 0.6 before and 4.1 ± 0.8 after) scores. In the responders, there was a significant decrease in the total Knodell score from 10 ± 0.6 to 7 ± 1 (P = 0.04), whereas the fibrosis stage (3 ± 0.6 before and 2.4 ± 0.8 after), as well as the activity score (6.6 ± 0.9 before and 5 ± 1.5 after), were stable. Conversely, in the non-responders, the fibrosis stage tended to increase from 1.3 ± 0.4 to 1.7 ± 0.5 (ns), whereas the total Knodell and activity scores did not significantly improve (6.4 ± 1.2 before versus 5.6 ± 1.1 after, and 5.1 ± 0.9 before versus 3.7 ± 1 after, respectively).

3.2. Biochemical response 4. Discussion At the beginning of the treatment, only 14 of the 18 patients had permanently increased levels of ALT and AST; conversely, gamma glutamyl transpeptidase (␥GT) levels were

Our study is the first conducted with HBV DNA(+) renaltransplant patients that shows the long-term efficacy and

Table 2 Virological parameters in 18 HBV DNA(+) renal transplant patients treated with lamivudine Patients

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total

HBV serology before 3TC/last FU

HBV DNA (log10 copies/ml)

HBs Ag

HBeAg

HBe Ab(+)

Before 3TC

Breakthrough (Y/N)

Last follow-up

+ + + + + + + + + + + + + + + + + + 18/18

+/− −/− +/+ −/− −/− −/− −/− +/+ −/− −/− −/− −/− −/− −/− +/+ −/− −/− −/− 4/18//3/18

−/+ +/+ −/− +/+ +/+ +/+ +/+ −/− +/+ +/+ +/+ +/+ +/+ +/+ −/− +/+ +/+ +/+ 14/18//15/18

8.3 8.6 7.1 7.9 7.9 9.6 8.6 7.9 9.01 8.1 7.6 7.9 9.6 7.9 8.3 9.4 9.3 9.6

N N N N N N Y Y Y Y Y Y N Y Y Y Y Y 11/18


HBV DNA polymerase mutation

Virological response

NA NA NA NA NA NA Y ND N Y N N N ND Y N Y ND

S S S S S S PR PR PR PR Rp Rp NR Rp Rp Rp Rp Rp

Abbreviations: NR: no virological response; PR: partial virological response; Rp: virological relapse following a virological response; S: sustained virological response; NA: not applicable; ND: not done.

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safety of lamivudine therapy. Despite a long period of treatment, there were no side-effects as far as renal and hematological parameters were concerned. HBV infection, although rare in chronic hemodialysis patients, might be severe after renal transplantation (Huang, 1997). Generally, HBV infection is ‘silent’ in dialysis patients (HBs Ag(+), normal ALT levels, low level of HBV DNA), whereas the onset of immunosuppression is associated with HBV reactivation. In this context, it has been shown that infection is detrimental in the long term, as far as the patient’s survival is concerned (Fornairon et al., 1996; Pfaff and Blanton, 1997). In the immunocompetent population, the first line of therapy for HBV infection relies on interferon alpha, and this gives good results (Wong et al., 1993). Those who do not respond to this treatment might receive lamivudine or adefovir. Lamivudine has been shown to be efficient at a daily dosage of 100 mg, i.e., less than the dosage used for HIV patients; this results in the inhibition of HBV replication and, in some patients, in seroconversion in the e-antigen system (Dienstag et al., 1995). In HBs Ag-positive renal transplant patients on lamivudine therapy Chan et al. have reported a rate of seroconversion in the e-antigen system of 21.4% (Chan et al., 2002). Meanwhile, in other patients, relapse may occur after withdrawal. To date, the long-term use of lamivudine has been associated with mutations in HBV DNA polymerase, up to 40.7% in renal transplant patients (Chan et al., 2002). It has already been reported that the likelihood of developing mutations in the HBV DNA polymerase gene was directly related to pre-therapeutic viral load, i.e., levels greater that 108 copies per ml (Mutimer et al., 1999; Puchhammer-Stockl et al., 2000). With lamivudine, mutations occur in the conserved subdomain C (YMDD motif), i.e., mutations of methionine 550 to either isoleucine (M550 I) or valine (M550 V). The M550 V mutation is almost always observed in conjunction with an additional mutation, L526 M, in conserved subdomain B (Bartholomeusz et al., 1998). However, it has been shown that these mutants remained sensitive, at least in vitro, to adefovir, an acyclic nucleotide monophosphate analogue (Xiong et al., 1998), or to other drugs (Ying et al., 2000). Recently, clinical trials using adefovir in both hepatitis B e-antigen(+) and negative replicative immunocompetent patients showed promising results (Hadziyannis et al., 2003; Marcellin et al., 2003). We observed that only four patients with breakthroughs during lamivudine therapy exhibited the mutations at positions L526 and M550 in HBV DNA polymerase. Therefore, the other patients might have had mutations at other positions, as already reported (Bartholomeusz et al., 1998). However, we are not sure if these mutations are of clinical relevance. Another hypothesis is that, due to the suboptimal concentration of lamivudine at cellular level, this might lower the selective pressure, thereby preventing the occurrence of mutants. In renal-transplant patients, interferon alpha therapy is associated with a high rate of acute rejection episodes (Rostaing et al., 1996). Ganciclovir was able to decrease the level of HBV DNA in transplant patients (Garnier et al., 1997), but

universal relapse occurs after withdrawal. Lamivudine therapy has been shown, at least in the short term, to be efficient in liver-transplant (Grellier et al., 1996; Kruger et al., 1996) as well as in renal-transplant patients (Jung et al., 1998; Rostaing et al., 1997). However, no long-term data are available. Moreover, in all these studies, HBV DNA was quantified by a non-ultrasensitive test that was only able to detect 106 copies/ml, i.e., 5 pg/ml, whereas ours was sensitive to as low as 400 copies/ml. In the present study, we have shown that, after lamivudine therapy withdrawal, a relapse always occurs in renal-transplant patients (Rostaing et al., 1997). We therefore resumed lamivudine, but only two patients responded again. Dienstag et al. (1995) found that, in HBV DNA(+) immunocompetent patients, only 50% of patients showed suppression of HBV DNA after 4 weeks of lamivudine at 100 mg/day compared with nearly 90% of patients treated with lamivudine at 300 mg/day. Therefore, if HBV DNA was still present at 3 months after the beginning of lamivudine therapy and when breakthrough occurred, we increased the dosage of lamivudine, sometimes up to 300 mg/day. In our study, with a mean follow-up of 36.5 months, using a sensitive test to quantify HBV DNA, only five of 18 patients are full responders as far as ALT and HBV DNA levels are concerned. However, eight patients, who initially had a full virological response, eventually relapsed despite increasing the daily dose of lamivudine. Conversely, we observed a virological response in one patient, at least as far as HBV DNA in the serum was concerned, and he experienced a mild increase in AST and ALT. This might be related to either a lack of sensitivity of our quantitative method in its assessment of HBV DNA, which is unlikely, or to a local immune response against HBV antigens, i.e., in the liver, because it is well known in hepatitis B virus infection that liver damage is mediated by an immune response (i.e., activity of the T lymphocytes). Altogether, these results are encouraging because, at the last follow-up, 10 patients (55%) still had a full or partial virological response. Although, not all the patients underwent an iterative liver biopsy, within 18–24 months of therapy their histology, overall, showed no change with respect to the stage of fibrosis or the activity score. In the virological responders, there was a statistically significant decrease in the Knodell score from 10 ± 0.6 to 7 ± 1 (P = 0.04). In the non-responder group, the decrease in activity score and the increase in fibrosis stage, although observed, were not statistically significant. This observation is very important if confirmed for a larger cohort of patients. Even if we did not clearly identify pre-therapeutic factors that might predict the virological response, we suggest that HBV DNA positive renal transplant patients should be treated in first instance with lamivudine; if there is no virological response or if virological breakthrough occurs during lamivudine therapy the patient can be switched to adefovir. However this latter drug has some degree of nephrotoxicity, and therefore might be used with cautious in patients already treated with anti-calcineurin agents.

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In conclusion, lamivudine therapy is safe in HBV(+)ve renal-transplant patients. However, even if, in the long term, the full and partial virological response rates are still high (55%), relapse or primary non-response do occur. The implementation of alternative efficient strategies is warranted, e.g., adefovir therapy.

Acknowledgment The authors thank Miss Dani`ele Mencia for her secretarial assistance.

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